Pericentromeric constitutive heterochromatin is a major part of the genome of humans and other eukaryotic organisms. Although it includes the centromere and other genetic elements and has many novel physical and genetic properties, the molecular structure and functional organization of heterochromatin is poorly understood owing to the relatively low density of genes and the abundance of repetitive DNA sequences. An important step toward the long range goal of physical mapping and sequencing the heterochromatin is the assembly of contigs covering large regions of heterochromatin. This has become possible using P1 clones in Drosophila. The project aims are to extend the existing P1 contigs, to generate new P1 contigs in selected regions of heterochromatin, and to exploit the existing contigs to study patterns and mechanisms of heterochromatin replication and underreplication in the Drosophila salivary gland chromosomes. Using STS-content mapping in combination with fluorescent in situ hybridization (FISH), the assembly of heterochromatic contigs will continue. Initial targets are to extend the existing large contigs that cover the euchromatin-heterochromatin junction of chromosome arm 3L, the Rsp-Bari region located deep in the heterochromatin of 2R, and the shorter contig containing the rolled gene also in 2R. New contigs will be generated around a novel sequence that hybridizes specifically with a small region near the centromere of chromosome 2 and around other suitable sequences. Relative to patterns of chromosome replication, a new method of quantitative PCR has been developed to estimate the level of replication of STS markers along a heterochromatic contig. A novel system for subcloning P1 clones will be employed to isolate subclones of average size 8-12 kb that span the transition zones between replication and underreplication. Further analysis of these subclones will be used to determine the specific DNA sequence motifs that delimit the regions of replication and underreplication. A candidate delimiter motif has already been identified. The continuity or discontinuity of the ends of the DNA duplexes in replicated regions will also be examined. The heterochromatic contigs and other data generated in this project will be important in the physical mapping and sequencing of heterochromatin and in further investigations of the molecular structure and function of heterochromatin.